Personnel
Christoph Cremer
University of Heidelberg
Kirchhoff-Institute for Physics
cremer@kip.uni-heidelberg.de
Research:
Quantitative molecular imaging analysis of specific nuclear genome structures
Far field optical light microscopy with its unique capability for contactless, non-destructive imaging inside thick transparent specimens such as cell nuclei has contributed widely to the present knowledge of the three-dimensional (3D-) architecture of the human genome in the cell nucleus. Visual microscopy observations and quantitative light optical analyses, especially confocal data, in combination with molecular labeling procedures, have revealed a highly structured nuclear genome organization suggesting the existence of "chromatin folding" and "chromatin positioning" codes.
A serious problem for the extension of "large scale" and "mesoscale" light microscopical studies of nuclear genome structure to nuclear genome nanostructure is the limited structural resolution. Due to the thickness of human cell nuclei in the order of several micrometer, far field methods have to be used if the cell is to remain three-dimensionally (3D) conserved. In recent years, various light optical approaches to overcome this impasse have been described. Examples are confocal laser scanning 4Pi-microscopy (pioneered by Stefan Hell, Goettingen/Heidelberg); micro-tomography, Spectral Precision Distance Microscopy (SPDM), or Spatially Modulated Illumination (SMI) microscopy (Kirchhoff Institute for Physics Heidelberg). These and other novel approaches allowed an increase in the optical, topological, and size resolution to the range of a few tens of nm (about 1/10 to 1/20 of the exciting visual light wavelength used). Their application will close the gap between the resolution achieved by ionizing radiation imaging and low photon energy microscopy.
A serious problem in the analysis of nuclear structures in the range of 100 nm and less is the disturbance of such structures by the molecular labelling procedure, especially denaturating ones. These effects can presently be overcome by "in vivo" labelling procedures like Green Fluorescent Protein labelling or replication labelling. In addition, novel "in vivo labelling" approaches like non-denaturating Fluorescent in situ Hybridization (FISH) of telomeres using PNA probes have been demonstrated . Another approach presently in development at University Heidelberg is Combinatorial Oligonucleotide FISH using purine/pyrimidine oligosequences.
Present State of Collaboration. In collaboration with Dr. Lindsay Shopland we have started to contribute to quantitative far field light optical analysis of nanostructures in mouse cell nuclei. Presently, the following methods are being established:
a) Quantitative image analysis of conventional light microscopical data;
b) Establishment of micro-tomography at IMB/TJL;
c) 4Pi-microscopy measurements of nuclear specimens prepared by Dr. Shopland; preliminary measurements have been performed using a 4Pi-Microscope at Leica Inc., Mannheim, presently the world’s most advanced commercially available highest resolution light microscope; such studies will be continued using Leica 4Pi-Microscopes which are being established both at IMB/TJL and the Kirchhoff-Institute for Physics (since April 2005).
d) Biocomputing of large-scale mouse nuclear genome structure and of the Piebald region. Presently, Brownian dynamics simulation of whole cell nuclei to determine motion patterns from entire chromosome territories to small gene cluster regions have been performed. The model allows a variety of experimentally testable predictions, such as apparent “diffusion” coefficients and gyration radii of chromosome territories and selected gene regions.
Future Research Plans. After establishment of a 4Pi confocal laser scanning microscope (“4Pi-Microscope) at the IMB/TJL, 4Pi-Microscopy studies of mouse nuclear genome structure will be performed directly at the IMB/TJL. Using individual cells deposited on special cover slides, such structures will be analyzed at increased optical resolution by two-photon excitation. In the 4Pi-Microscopy One Photon Excitation mode, topological analysis using Spectral Precision Distance (SPDM) microscopy will be performed at an increased precision compared to conventional confocal microscopy.
Because of the problem of preservation of nuclear nanostructures, we shall first concentrate either on specimens not requiring conventional FISH procedures (in the high resolution range beyond 100 nm); or using denaturating molecular labelling procedures, we shall focus on questions in the 100 nm regime.
In collaboration with the Kirchhoff-Institute Heidelberg, we plan to use SMI-microscopy and SPDM in comparative studies of the same regions if possible in the same cells. For this, we are presently developing a collaboration with Dr. Joachim Spatz a novel cell positioning system based on fluorescent surface nanostructures.
We will explore using the 4Pi-Microscope for the study of tissues/embryos up to a thickness in the hundred to several hundred micrometer range at conventional optical resolution (but at doubled axial specimen extension compared to conventional confocal microscopy).
Together with Dr. Jay Nadeau and Dr. Netta Cohen, we plan to develop novel methods for "in vivo" molecular labeling of specific nuclear genome nanostructures by oligonucleotides attached to fluorescent nanoparticles in combination with nuclear localization signals.
Recent Publications:
Kreth G, Finsterle J, Cremer C. 2004. Virtual radiation biophysics: implications of nuclear structure. Cytogenet Genome Res, (in press).
Kreth G, Finsterle J, Hase JV, Cremer M, Cremer C. 2004. Radial arrangement of chromosome territories in human cell nuclei: a computer model approach based on gene density indicates a probabilistic global positioning code. Biophys J, (in press.)
Spöri U, Failla AV, Cremer C. 2004. Superresolution size determination in fluorescence microscopy: A comparison between spatially modulated illumination and confocal laser scanning microscopy, (submitted).
Martin S, Failla AV, Spöri U, Cremer C, Pombo A. 2004. Measuring the size of biological nanostructures with spatially modulated illumination microscopy, (submitted).
Schweitzer A, Eipel H, Cremer C. 2004. Rapid Image Acquisition in Multi-Photon Excitation Fluorescence Microscopy, (submitted).
Kreth G, Finsterle J, von Hase J, Cremer M, Cremer C. 2004. Radial arrangement of chromosome territories in human cell nuclei: A computer model approach based on gene density indicates a probabilistic global positioning code. Biophys J 86:2803–2812.
Cremer M, Zinner R, Stein S, Albiez H, Wagler B, Cremer C, Cremer T. 2004. Three dimensional analysis of histone methylation patterns in normal and tumor cell nuclei. Eur J Histochem 48:11-23.
Martin S, Failla AV, Spöri U, Cremer C, Pombo A. 2004. Measuring the size of biological nanostructures with spatially modulated illumination microscopy. Mol Biol Cell 15:2449–2455.
Bolzer, Kreth G, Solovei I, Koehler D, Saracoglu K, Fauth C, Müller S, Eils R, Cremer C, Speicher MR, Cremer T. 2005. Three-dimensional maps of all chromosomes in human male fibroblast nuclei and prometaphase rosettes. PLoS Biology 3:1-17.
Hildenbrand G, Rapp A, Spoeri U, Wagner Ch, Cremer C, Hausmann M. 2005. Nano-sizing of specific gene domains in intact human cell nuclei by Spatially Modulated Illumination (SMI) light microscopy. Biophysis J 88:4312-4318.
Wagner C, Spoeri U, Cremer C. 2005. High-precision SMI microscopy size measurements by simultaneous frequency domain reconstruction of the axial point spread function. Optik 116:15–21.
Mathee H, Baddeley D, Wotzlaw C, Fandrey J, Cremer C, Birk U. 2005. Nanostructure of specific chromatin regions and nuclear complexes. Histochem Cell Biol, (in press).
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